Apoptosis is a form of cell death orchestrated by chains of biochemical reactions. Cells undergoing apoptosis show characteristic morphological features such as condensation of cytoplasmic and nuclear contents, blebbing of plasma membranes, fragmentation of nuclei, and ultimately breakdown into membrane-bound apoptotic bodies that are rapidly phagocytosed (Kerr et al., 1972).
Mitochondria play an important role in regulating apoptosis induced by intracellular damaging signals such as DNA damage in mammalian cells (Danial and Korsmeyer, 2004). Apoptotic stimuli exert their effects on mitochondria to cause the release of pro-apoptotic factors like cytochrome c and Smac/Diablo. These factors either directly activate caspases, a group of intracellular cysteine proteases that execute apoptosis by cleaving their substrates, or release caspase-inhibition imposed by the inhibitor of apoptosis proteins (IAPs) (Du et al., 2000; Liu et al., 1996; Verhagen et al., 2000).
Mitochondrial response to apoptotic stimuli is regulated by the pro- and anti-apoptotic Bcl-2 family of proteins (Gross et al., 1999; Martinou and Green, 2001). Anti-apoptotic proteins such as Bcl-2, Bcl-xL, and Mcl-1 protect mitochondrial integrity, while the pro-apoptotic members of the family promote the release of apoptogenic proteins from mitochondria. The fate of a cell under apoptotic stimulation is determined by the balance of the function between the pro- and anti-apoptotic members of Bcl-2 family. Therefore, studying the upstream regulation of Bcl-2 family proteins will be critically important for our understanding of apoptosis regulation.
Among the anti-apoptotic members of the Bcl-2 family proteins, Mcl-1 is unique in that it is an early-response gene that can be rapidly induced and turned over (Kozopas et al., 1993; Yang et al., 1996; Yang et al., 1995). This property enables Mcl-1 to function at an apical step in a signaling cascade consisting of Bcl-2 family proteins and provides an acute protective function against apoptosis induced by a variety of stimuli including DNA damage, adenoviral infection, growth factors withdrawal and treatment of cytotoxic agents (Cuconati et al., 2003; Derouet et al., 2004; Huang et al., 2000; Le Gouill et al., 2004; Nijhawan et al., 2003; Piret et al., 2004; Zhang et al., 2002; Zhou et al., 1998; Zhou et al., 1997). Consistently, disappearance of Mcl-1 is associated with the onset of apoptosis and is achieved by the combination of synthesis blockage and continuous degradation (Cuconati et al., 2003; Nijhawan et al., 2003).
The degradation of Mcl-1 in HeLa cells can be blocked by proteasome inhibitors, suggesting a role for the ubiquitin proteasome pathway in apoptosis upstream of Bcl-2 family of proteins (Derouet et al., 2004; Nencioni et al., 2004; Cuconati et al., 2003; Nijhawan et al., 2003). Here, we identify an E3 ubiquitin ligase that mediates the polyubiquitination of Mcl-1. This protein is a novel HECT-domain containing ubiquitin ligase, which we named Mule for Mcl-1 ubiquitin ligase E3. Mule is the first E3 ligase specifically identified that regulates the Bcl-2 family of proteins. The carboxy 308 amino acids of human Mule are identical to a protein previously identified as a 308 amino acid named UREB1 for “upstream regulatory element binding protein 1” (Gu et al, 1994; GenBank ID 3694922); and amino acids 1055-4374 of Mule are identical to amino acids 40-3360 of gi: 22090626, which entered GenBank August, 2002, and is named LASU1 for large structure of UREB1.
We made a presentation at the 2003 Cold Spring Harbor meeting on programmed cell death about the identification and purification of Mule without revealing its identity other than its name and that it was a large, HECT-domain containing protein (Meier P., Silke J., 2003).
Subsequent to our identification of Mule, the same protein was reported as functioning as an E3 enzyme that ubiquitinates histones in spermatids during spermatogenesis (Liu et al, 2005). The human and mouse Mule polypeptide sequences are listed in GenBank as gi:61676188 (accession no. NP—113584.3) and gi:61676190 (accession no. NP—067498.3), respectively, and the corresponding nucleotide sequences are gi:61676187 (accession no. NM—031407.3) and 61676189 (accession no. NM—21523), respectively.